Project Summary/Abstract Dopamine receptors are the primary therapeutic targets for a variety of neurological and psychiatric disorders, including schizophrenia and Parkinson's disease. The five subtypes of dopamine receptors (D1- D5) are members of the superfamily of G protein-coupled receptors. The D1 and D2 subtypes are the most abundant and mediate most classic dopamine-dependent behaviors. The overall long-term goal of this research program is to delineate and distinguish between D1 and D2 receptor mechanisms, from the initial step of ligand binding to the receptor to the final manifestation of dopamine-dependent behaviors. A secondary aim is to assess the differential functional roles of the alternatively spliced forms of the D2 receptor, D2S and D2L. The first specific aim is driven by the hypothesis that D1 receptor agonists, but not D2 agonists, must have a catechol ring because of distinct residues at two positions in transmembrane helices 6 and 7. We will characterize reciprocal mutants in which one or both of these residues in one receptor subtype is changed to the corresponding residue(s) of the other subtype. We will determine the effect of the mutations on high- and low-affinity binding of and regulation of cyclic AMP accumulation by catecholamine and non-catecholamine agonists. The second specific aim is based on the hypothesis that activation of D1 and D2 receptors regulates their localization in lipid rafts and differentially regulates the abundance of other raft-enriched proteins. We propose to examine the distribution of the receptors in non- raft fractions and the effect of acute or repeated agonist treatment on receptor distribution. In addition, proteomic analyses of raft fractions will be used to identify drug-induced changes in other raft proteins. The third specific aim will test the hypotheses that D2L is the postsynaptic D2 receptor that mediates increased locomotor activity and D2S is the presynaptic autoreceptor that regulates dopamine release and tyrosine hydroxylase activity in the mouse basal forebrain. We will evaluate the behavioral and neurochemical effects of HSV-mediated expression of D2L or D2S in the nucleus accumbens (postsynaptic) or ventral tegmental area (presynaptic) of D2 receptor null-mutant mice. The fourth specific aim is driven by the hypotheses that S100B binding to the D2 receptor is necessary for most efficient function, and that there are D1 and D2 receptor-interacting proteins still to be identified that regulate dopamine receptor signaling and localization. We will carry out additional rounds of cDNA library screening with D1 and D2 receptor intracellular loops to identify new dopamine receptor-interacting proteins and investigate the consequences of the binding of these proteins and S100B to dopamine receptors for the subcellular localization and signaling properties of the receptors. We hope that, by identifying structural features of D1 and D2 receptors that interact with ligands in a subtype-selective manner to determine binding and activation of signaling pathways, dissecting the contributions of dopamine receptor subtypes to biochemical and behavioral responses to dopamine and synthetic agonists, and characterizing novel proteins that regulate dopamine receptor signaling, these studies will contribute to the development of more selective pharmaceutical treatments of neuropsychiatric disorders that involve the dopamine system. PUBLIC HEALTH RELEVANCE: Project Narrative Each year, VA Medical Centers hospitalize about 45,000 veterans for treatment of schizophrenia and treat at least 40,000 Parkinson's disease patients. The causes of the diseases are unknown, but changes in the density of dopamine D2-like receptors have been implicated in the pathophysiology of the schizophrenia, and the primary treatments for the diseases are blockade or stimulation of dopamine receptors. Analysis of endogenous and recombinant dopamine receptors expressed in neurons and in mammalian cell lines, and of the function of dopamine receptors in the behaving rodent, are powerful model systems with which to perform basic research that should contribute to the development of rational strategies for improved treatment of schizophrenia and movement disorders.